A simple, rapid, and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method for the measurement of mupirocin concentrations in both skin layers and percutaneous samples has been developed. Mupirocin was extracted from skin layers using PBS–acetonitrile (90:10, v/v). The method is sufficiently sensitive and repeatable to be used in percutaneous penetration studies. The samples were chromatographed on a View the MathML source mm C8 LiChrospher Select B (5 μm). The mobile phase composition was a mixture of acetonitrile–ammonium acetate 0.05 M (27.5:72.5, v/v) adjusted to pH 6.3 with acetic acid. The analyte was detected at 228 nm and the run time was 11 min. Linearity was confirmed in the concentration range 0.2–20 μg/ml and the limit of detection was 9.5 ng/ml.